Methods for amplifying nucleic acids on substrates

ABSTRACT

A method is provided herein, the method includes: applying a sample comprising target nucleic acids to a sample application zone of a substrate; applying an aqueous buffer to the sample application zone of the substrate to washes away one or more inhibitors present on the sample application zone; and applying an isothermal nucleic acid amplification reaction mixture to the sample application zone to amplify the target nucleic acid to form a nucleic acid amplification product. The target nucleic acid having a first molecular weight is substantially immobilized at the sample application zone and wherein the amplification product having a second molecular weight.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 23, 2014, isnamed 276639-1_SL.txt and is 4,742 bytes in size.

FIELD

The invention generally relates to methods and substrates for amplifyingtarget nucleic acids.

BACKGROUND

Preparation and manipulation of high quality nucleic acids are primaryrequirements for a variety of applications, such as analyte-detection,sensing, forensic and diagnostic applications, genome sequencing, andthe like. Various applications of nucleic acids are typically precededby a purification process to eliminate unwanted contaminants from thenucleic acids, which may interfere in downstream applications.Techniques including gel electrophoresis, capillary electrophoresis orelectrophoresis in microfluidic or microanalytical devices, which aremainstay in molecular and cell biology and enable purification andseparation of specific nucleic acids.

The separation of nucleic acids provide information on size of thenucleic acids, which is useful for predicting a number of geneticdisorders, such as genetic pre-dispositions or acquired mutations/localrearrangements for deoxyribonucleic acid (DNA). The ribonucleic acid(RNA) profiles represent “snap shots” of the cell's biology, since theyare continuously changing in response to the surrounding environment.

In some applications, nucleic acid analysis requires sample-preparationinvolving multiple steps, such as collection, separation or purificationof the nucleic acids from a biological sample. A simplified method forpreparing nucleic acid sample for subsequent analysis is highlydesirable. A simultaneous separation and amplification of nucleic acidsis especially required when the quantity of the biological sample isless, for example, the sample procured for biopsy or a sample collectedfor forensic application.

Different technologies have been developed to separate nucleic acidsfrom a liquid sample using a substrate, which includes: separatingnucleic acids from a sample by flowing the sample along a bibulousmembrane to distribute along the length of the membrane. In anothermethod, at least two cellular components (such as, genomic DNA, RNA andproteins) is separated, wherein an aqueous solution including thecellular components applied to multiple mineral supports followed bywashing. In many of these methods, the substrate requires a washing stepwith a buffer or a solution, which is not compatible with the subsequentprocess steps. The washing buffer needs to remove from the substratebefore executing the subsequent steps. These methods are time consumingand complex as they require multiple steps (such as washing or elution)or multiple substrates.

The increased use of nucleic acids requires fast, simple and reliablemethods and systems for amplifying and separating nucleic acids.

BRIEF DESCRIPTION

In one embodiment, a method comprises applying a sample comprisingtarget nucleic acids to a sample application zone of a substrate;applying an aqueous buffer to the sample application zone of thesubstrate to wash away or dilute one or more inhibitors present on thesample application zone; and applying an isothermal nucleic acidamplification reaction mixture to the sample application zone to amplifythe target nucleic acid to form a nucleic acid amplification product;wherein the target nucleic acid having a first molecular weight issubstantially immobilized at the sample application zone and theamplification product has a second molecular weight.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a schematic diagram of a device in accordance with anexample of an embodiment of the invention.

FIG. 2 illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 3 illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 4 illustrates a schematic diagram of a device in accordance with anexample of an embodiment of the invention.

FIG. 5A illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 5B illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 5C illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 5D illustrates a schematic diagram of a device in accordance withanother example of an embodiment of the invention.

FIG. 6 illustrates top view (top) and side view (bottom) of a schematicdiagram of a substrate in accordance with one embodiment of theinvention.

FIG. 7A illustrates side view of a schematic diagram of an example of anitrocellulose substrate (NC) and a sample application zone (FTA) on thesubstrate in accordance with one embodiment of the invention.

FIG. 7B illustrates an FTIR-ATR spectroscopy for different substratecomposition to determine residual composition on washing.

FIG. 8A is a gel electrophoresis image showing bands for amplificationproducts of genomic DNA applied in solution, on the substrate after washwith aqueous buffer and on the substrate without wash, compared tonon-template control using Ping Pong amplification.

FIG. 8B is a gel electrophoresis image showing bands for amplificationproducts of genomic DNA applied in solution, on the substrate after washwith aqueous buffer and on the substrate without wash, compared tonon-template control using UStar amplification.

FIG. 8C is a gel electrophoresis image showing bands for amplificationproducts of genomic DNA applied in solution, on the substrate after washwith aqueous buffer and on the substrate without wash, compared tonon-template control using helicase dependent amplification (HDA)amplification.

FIG. 9A is a flow diagram that illustrates sample application, washingand cutting the substrate in pieces for analyzing by gelelectrophoresis.

FIG. 9B illustrates a gel electrophoresis image to determine lateralflow of genomic DNA obtained in the flow diagram of FIG. 9A, example 6.

FIG. 10 A is a flow diagram that illustrates sample application,amplification and cutting the substrate into pieces for analyzing by gelelectrophoresis.

FIG. 10 B illustrates an electrophoresis image to determine lateral flowof target DNA and amplicons obtained in a flow diagram of FIG. 10A,example 7.

FIG. 10 C illustrates a gel electrophoresis image to determine a lateralflow of non-template control (NTC) obtained in flow diagram of FIG. 10A,example 7.

FIG. 11 A is a flow diagram that illustrates sample application,amplification on lateral flow of amplification reagent, and detection onlateral flow of detection probes on the substrate.

FIG. 11 B is an image that illustrates detection of amplicons whencompared with a substrate with no cell.

DETAILED DESCRIPTION

Various embodiments provide suitable methods and substrates forextraction of nucleic acids from a biological sample, followed byamplification and separation of the nucleic acid amplicons from eachother and/or from unwanted contaminants based on molecular weights ofdifferent nucleic acid (amplicon) species or contaminants. The substrateis configured to collect a biological sample, extract nucleic acids fromthe sample followed by amplification and separation on the samesubstrate. The eluted nucleic acids are used in various downstreamanalysis or applications.

To more clearly and concisely describe the subject matter of the claimedinvention, the following definitions are provided for specific terms,which are used in the following description and the appended claims.Throughout the specification, exemplification of specific terms shouldbe considered as non-limiting examples.

The singular forms “a”, “an” and “the” include plural referents unlessthe context clearly dictates otherwise. Approximating language, as usedherein throughout the specification and claims, may be applied to modifyany quantitative representation that could permissibly vary withoutresulting in a change in the basic function to which it is related.Accordingly, a value modified by a term such as “about” is not to belimited to the precise value specified. In some instances, theapproximating language may correspond to the precision of an instrumentfor measuring the value. Where necessary, ranges have been supplied, andthose ranges are inclusive of all sub-ranges there between.

The term “nucleic acid” as referred to herein comprises all forms of DNA(e.g. genomic DNA, mtDNA) or RNA (mRNA, tRNA, rRNA, small RNA, siRNA,miRNA, non-coding RNA, animal RNA, plant RNA, viral RNA or bacterialRNA), as well as recombinant RNA and DNA molecules or analogues of DNAor RNA generated using nucleotide analogues. The nucleic acids may besingle stranded or double stranded. The nucleic acids may include thecoding or non-coding strands. The term also comprises fragments ofnucleic acids, such as naturally occurring RNA or DNA which may berecovered using the extraction methods disclosed. Nucleic acid may alsorefer to a portion of a nucleic acid (e.g., RNA or DNA). The extractednucleic acids may further comprise peptide nucleic acids (PNA).

Separated nucleic acids may comprise single type of nucleic acids or twoor more different types of nucleic acids. The nucleic acids may besingle-stranded, double-stranded, linear or circular. Molecular weightsof separated nucleic acids are also not limited, may be optional in arange from several base pairs (bp) to several mega base pair (Mbp).

As used herein, the term “target nucleic acid” refers to a nucleic acid(such as DNA or RNA) sequence of either natural or synthetic origin thatis desired to be amplified in an amplification reaction. The targetnucleic acid may be obtained from a biological sample in vivo or invitro. For example, the target nucleic acid may be obtained from abodily fluid (e.g., blood, blood plasma, serum, or urine), an organ, atissue, a cell, a sectional portion of an organ or tissue, a cellisolated from a biological subject (e.g., a region containing diseasedcells, or circulating tumor cells), a forensic sample or an ancientsample. The biological sample that contains, or is suspected to contain,the target nucleic acid may be of eukaryotic origin, prokaryotic origin,viral origin or bacteriophage origin. For example, the target nucleicacid may be obtained from an insect, a protozoa, a bird, a fish, areptile, a mammal (e.g., rat, mouse, cow, dog, guinea pig, or rabbit),or a primate (e.g., chimpanzee or human). The target nucleic acid mayalso be a complementary DNA (cDNA) that is generated from an RNAtemplate (e.g., mRNA, ribosomal RNA) using a reverse transcriptaseenzyme. A DNA product generated by another reaction, such as a ligationreaction, a PCR reaction, or a synthetic DNA may also serve as asuitable target nucleic acid. The target nucleic acid may be dispersedin solution or may be immobilized on a solid support, such as in blots,arrays, glass slides, microtiter plates, beads or ELISA plates. A targetDNA or the entire region of a target DNA may be amplified by a DNApolymerase in a DNA amplification reaction to produce amplificationproducts or amplicons.

As used herein, the term “sample application zone” refers to an area ona substrate, wherein a sample is applied to that area or zone of thesubstrate for further processing. The sample application zone is a partof the same substrate. In some embodiments the sample application zonemay comprise impregnated reagents, such as stabilizing reagents or celllysis reagents. The sample application zone may be a paper comprisingreagents disposed on the substrate.

As used herein, the term “detection zone” refers to an area on asubstrate, wherein the nucleic acids of a sample is separated accordingto its molecular weight at the detection zone of the substrate. Thedetection zone is a part of the same substrate. In some embodiments, thedetection zone may comprise impregnated reagents, such as stabilizingreagents or buffer reagents. The detection zone may be a papercomprising reagents disposed on the substrate.

“Amplicons” or “amplification product” may include multiple copies ofthe target nucleic acid or multiple copies of sequences that arecomplementary to the target nucleic acid. The target nucleic acid, suchas DNA acts as a template in a DNA amplification reaction to produceamplicons. Either a portion of a target DNA or the entire region of atarget DNA may be amplified by a DNA polymerase in a DNA amplificationreaction to produce amplification products or amplicons.

As used herein, the term “substantially immobilized” refers to aquantity of nucleic acids having certain molecular weights, which arepositioned around a particular positioning portion. The immobilizationof the nucleic acids may occur due to higher molecular weight of thenucleic acids. The nucleic acids having higher molecular weighttypically have lower mobility while flowing a buffer along the length ofthe substrate. The substantial quantity of nucleic acids may berepresented as the percentage of the total amount of nucleic acidshaving a particular molecular weights in the sample solution immobilizeat a particular position. For example, substantially the nucleic acidswith first molecular weight means 90% of the total target nucleic acidsapplied to the substrate immobilized at the substrate at or around thesample application zone.

As used herein the term “oligonucleotide” refers to an oligomer ofnucleotides. A nucleotide may be represented by its letter designationusing alphabetical letters corresponding to its nucleoside. For example,A denotes adenosine, C denotes cytidine, G denotes guanosine, U denotesuridine, and T denotes thymidine (5-methyl uridine). W denotes either Aor T/U, and S denotes either G or C. N represents a random nucleosideand may be any of A, C, G, or T/U. A star (*) sign preceding a letterdesignation denotes that the nucleotide designated by the letter is aphosphorothioate-modified nucleotide. For example, *N represents aphosphorothioate-modified random nucleotide. A plus (+) sign preceding aletter designation denotes that the nucleotide designated by the letteris a locked nucleic acid (LNA) nucleotide. For example, +A represents anadenosine LNA nucleotide, and +N represents a locked random nucleotide.The oligonucleotide may be a DNA oligonucleotide, an RNA oligonucleotideor a DNA-RNA chimeric sequence. Whenever an oligonucleotide isrepresented by a sequence of letters, the nucleotides are in 5′→3′ orderfrom left to right. For example, an oligonucleotide represented by aletter sequence (W)_(x)(N)_(y)(S)_(z), wherein x=2, y=3 and z=1,represents an oligonucleotide sequence WWNNNS, wherein W is the 5′terminal nucleotide and S is the 3′ terminal nucleotide (“Terminalnucleotide” refers to a nucleotide that is located at a terminalposition of an oligonucleotide sequence. The terminal nucleotide that islocated at a 3′ terminal position is referred as a 3′ terminalnucleotide, and the terminal nucleotide that is located at a 5′ terminalposition is referred as a 5′ terminal nucleotide).

As used herein the dNTP mixture refers to a mixture deoxyribonucleosidetriphosphates, where N is a random nucleotide including any of A, C, G,or T/U.

As used herein, “primer”, or “primer sequence” refers to a short linearoligonucleotide that hybridizes to a target nucleic acid sequence (e.g.,a deoxyribonucleic acid (DNA)) to prime a nucleic acid amplificationreaction. The primer may be a ribonucleic acid (RNA) oligonucleotide, aDNA oligonucleotide, or a chimeric sequence. The primer may containnatural, synthetic, or modified nucleotides. Both the upper and lowerlimits of the length of the primer are empirically determined. The lowerlimit on primer length is the minimum length that is required to form astable duplex upon hybridization with the target nucleic acid undernucleic acid amplification reaction conditions. Very short primers(usually less than 3-4 nucleotides long) do not form thermodynamicallystable duplexes with target nucleic acids under such hybridizationconditions. The upper limit is often determined by the possibility ofhaving a duplex formation in a region other than the pre-determinednucleic acids sequences in the target nucleic acids. As a non-limitingexample, suitable primer lengths are often in the range of about 4 toabout 40 nucleotides long. A primer may also be used to capture anucleic acid sequence.

As used herein, the terms “amplification”, “nucleic acid amplification”,or “amplifying” refer to the production of multiple copies of a nucleicacid template, or the production of multiple nucleic acid sequencecopies that are complementary to the nucleic acid template.

As used herein, “multiple displacement amplification” (MDA) refers to anucleic acid amplification method, wherein the amplification involvesthe steps of annealing multiple primers to a denatured nucleic acidfollowed by DNA synthesis in which downstream double stranded DNAregion(s) which would block continued synthesis is disrupted by a stranddisplacement nucleic acid synthesis through these regions. As synthesisproceeds through an area of double stranded DNA, and the synthesis ofthe new strand occurs while displacing the existing strand, there is anet increase in that sequence of DNA, or DNA amplification. This istermed as “strand displacement amplification”, which occurs when onestrand is displaced by the synthesis of a new strand. As nucleic acid issynthesized by strand displacement, single stranded DNA is generated bythe strand displacement, and as a result, a gradually increasing numberof priming events occur, forming a network of hyper-branched nucleicacid structures. MDA is highly useful for whole-genome amplification forgenerating high-molecular weight DNA from a small amount of genomic DNAsample with limited sequence bias. Any strand displacing nucleic acidpolymerase that has a strand displacement activity apart from itsnucleic acid synthesis activity (e.g., Phi29 DNA polymerase or a largefragment of the Bst DNA polymerase) may be used in MDA. MDA is oftenperformed under isothermal reaction conditions, using random primers forachieving amplification with limited sequence bias.

As used herein, the term “rolling circle amplification (RCA)” refers toa nucleic acid amplification reaction that amplifies a circular nucleicacid template (e.g., single stranded DNA circles) via a rolling circlemechanism. RCA is initiated by the hybridization of a primer to acircular, often single-stranded, nucleic acid template. The nucleic acidpolymerase then extends the primer that is hybridized to the circularnucleic acid template by continuously progressing around the circularnucleic acid template to replicate the sequence of the nucleic acidtemplate over and over again (rolling circle mechanism). RCA typicallyproduces concatamers comprising tandem repeat units of the circularnucleic acid template sequence complement.

As used herein, the term “Ping Pong amplification” refers to a nucleicacid amplification reaction that amplifies a nucleic acid template usingmultiple primers, which accelerate the amplification process. A pair ofprimers comprising a forward primer and a reverse primer may be used fortwo template strands, such as a plus strand ((+) strand) and a minusstrand ((−) strand) for an amplification reaction. A reverse primer maybe a primer that anneals to a complementary (+) strand to furthergenerate a (−) strand in the reverse direction. For example, when areverse primer anneals to the complementary strand of target DNA at adefined distance from the forward primer, amplification process isaccelerated. Since the targets for each of these primers would bepresent in the original template, both strands would be amplified in thetwo primer scheme. The reaction is referred as “Ping-Pong” reaction,wherein the “Ping product” is the amplicon of the forward primer and the“Pong product” is the amplicon of the reverse primer. The inclusion ofmultiple paired primers may improve the relative percentage of adiscrete product in the reaction mixture.

As used herein, the term “Helicase-dependent amplification (HDA)” refersto an isothermal amplification reaction that utilizes a DNA helicase.The double stranded DNA separates to form single-stranded templates forprimer hybridization by DNA helicase and subsequently primer extensionis achieved by a DNA polymerase.

As used herein, the term “DNA polymerase” refers to an enzyme thatsynthesizes a DNA strand de novo using a nucleic acid strand as atemplate. DNA polymerase uses an existing DNA or RNA as the template forDNA synthesis and catalyzes the polymerization of deoxyribonucleotidesalongside the template strand, which it reads. The newly synthesized DNAstrand is complementary to the template strand. DNA polymerase can addfree nucleotides only to the 3′-hydroxyl end of the newly formingstrand. It synthesizes oligonucleotides via transfer of a nucleosidemonophosphate from a deoxyribonucleoside triphosphate (dNTP) to the3′-hydroxyl group of a growing oligonucleotide chain. This results inelongation of the new strand in a 5′→3′ direction. Since DNA polymerasecan only add a nucleotide onto a pre-existing 3′-OH group, to begin aDNA synthesis reaction, the DNA polymerase needs a primer to which itcan add the first nucleotide. Suitable primers comprise oligonucleotidesof RNA or DNA or nucleotide analogs. The DNA polymerases may be anaturally occurring DNA polymerases or a variant of natural enzymehaving the above-mentioned activity. For example, it may include a DNApolymerase having a strand displacement activity, a DNA polymeraselacking 5′→3′ exonuclease activity, a DNA polymerase having a reversetranscriptase activity, or a DNA polymerase having an exonucleaseactivity.

As used herein, the terms “strand displacing nucleic acid polymerase” or“a polymerase having strand displacement activity” refer to a nucleicacid polymerase that has a strand displacement activity apart from itsnucleic acid synthesis activity. A strand displacing nucleic acidpolymerase can continue nucleic acid synthesis on the basis of thesequence of a nucleic acid template strand by reading the templatestrand while displacing a complementary strand that is annealed to thetemplate strand. The strand displacing nucleic acid polymerase includesDNA polymerase, RNA polymerase, and reverse transcriptase.

The term, “reducing agents” as referred to herein include any chemicalspecies that provides electrons to another chemical species. A varietyof reducing agents are known in the art. Examples of reducing agentsinclude dithiothreitol (DTT), 2-mercaptoethanol (2-ME), andtris(2-carboxyethyl)phosphine (TCEP). Moreover, any combination of theseor other reducing agents may be used. In particular embodiments, thereducing agent is TCEP.

The term “amplification buffer” as used herein includes, but is notlimited to, 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris),2-(N-morpholino) ethanesulfonic acid (MES),3-(N-morpholino)propanesulfonic acid (MOPS), citrate buffers,4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), andphosphate buffers. The amplification buffer further includes, forexample, Tris-HCl, diammonium sulphate, monovalent cation (such as KCl),divalent cation (such as MgSO₄) or Tween®20. This list of potentialbuffers is for illustrative purposes only. The pH of the buffer istypically titrated in the range of 6 to 8. In some embodiments, thebuffer comprises dNTPs, BSA or combination thereof.

The term “separate, separating or separation” used herein indicates theact or action to isolate or purify nucleic acids from unwantedcontaminants of a sample solution and/or from each other according tomolecular weights.

The term “biological sample” is used in a broad sense and is intended toinclude a variety of physiological or clinical biological sources thatinclude nucleic acids. Such sources include, without limitation, wholetissues, including biopsy materials and aspirates; in vitro culturedcells, including primary and secondary cells, transformed cell lines,and tissue and blood cells; body fluids such as urine, sputum, semen,secretions, eye washes and aspirates, lung washes and aspirates; mediafrom DNA or RNA synthesis; mixtures of chemically or biochemicallysynthesized DNA or RNA; fungal and plant tissues, such as leaves, roots,stems, and caps; microorganisms and viruses that may be present on or ina biological sample; bacterial cells; and any other source in which DNAand/or RNA is or may be in.

The sample solution is a solution comprising either or both of DNA andRNA, or, cells, cell components or cell extracts which comprise eitheror both of DNA and RNA, dissolved, suspended, mixed or otherwiseincluded therein. The sample solution may be a solution prepared from abiological sample.

One or more embodiments of a method are provided, wherein the methodcomprises applying a sample comprising target nucleic acids to a sampleapplication zone of a substrate. An aqueous buffer may further beapplied to the sample application zone for washing away or diluting oneor more inhibitors from the sample application zone. The washing isfollowed by applying an isothermal nucleic acid amplification reactionmixture to the sample application zone of the substrate to amplify thetarget nucleic acid to form a nucleic acid amplification product oramplicon. The target nucleic acid having a first molecular weight issubstantially immobilized at the sample application zone and theamplification product has a second molecular weight.

An aqueous buffer may be applied at the sample application zone forwashing the substrate. Non-limiting examples of the term “applied”,“apply” or “applying” include, contacting, adding or disposing a sampleor an amplification reagent or a washing solution (such as aqueousbuffer) on the substrate using a tube, pipette, catheter, syringe,conduit, an automatic injector, or using any other applicableways/tools. In some embodiments, the sample may be poured onto thesubstrate. The term “apply” may also include flowing the aqueous buffer,amplification buffer or amplification reagent including a polymerasethrough the substrate.

In these embodiments, the substrate may be of any shape or size. Thesubstrate may be rectangular, square planar, circular, elliptical orirregular in shape. In one embodiment, the substrate is circular,wherein the sample application zone may be at the center of the circularsubstrate. In some embodiments, the substrate is rectangular shape, forexample the substrate is a longitudinal strip, wherein the sampleapplication zone is at one end of the substrate.

In one example, the substrate is a longitudinal strip, wherein anaqueous buffer is flowed through the strip to wash away or dilute theinhibitors. In some embodiments, the substrate may be washed or dilutedby dipping the substrate in an aqueous buffer or incubating thesubstrate in an aqueous buffer for some time. The isothermalamplification reaction mixture may be applied to the sample applicationzone after washing the substrate.

In some other embodiments of a method, the method comprises applying asample comprising target nucleic acids to a sample application zone of asubstrate; and flowing a nucleic acid amplification reaction mixtureacross a length of the substrate through the sample application zone toamplify the target nucleic acid to form a nucleic acid amplificationproduct or amplicon. The target nucleic acid having a first molecularweight is substantially immobilized at the sample application zone andthe amplification product having a second molecular weight migrates awayfrom the sample application zone.

As noted, the sample is applied to a sample application zone of thesubstrate, which may be present at either end of the substrate. Todescribe the method steps sufficiently, the substrate design is brieflydescribed herein to generally correlate the method steps to the devicecomponents. Referring to FIG. 1, a device 10, in accordance with oneembodiment, comprises a substrate 12, with a first end 11 and a secondend 31. The substrate is also shown in FIG. 6. The device 10 furthercomprises an amplification reagent reservoir 24, a wash reagentreservoir 26, an amplification reagent wicking pad 28 and a wash reagentwicking pad 30.

The substrate 12 comprises a sample application zone 14, which may alsobe used as a sample lysis zone and/or nucleic acid stabilization zone.The substrate further comprises fuses 16, which restrain the sample tobe located at the sample application zone 14. The substrate furthercomprises a detection zone 20, which is located at the opposite end ofthe sample application zone 14, and near to the second end 31 of thesubstrate. In one or more embodiments, the substrate further comprisesan amplification zone heating unit 22 covering the whole longitudinalstrip. The substrate optionally contains a heating unit at or near thesample application zone for drying the loaded sample.

The method comprises applying a sample comprising target nucleic acidsto a sample application zone of a substrate; wherein the substrate is anelongated strip 12. The sample solution, amplification reagent orwashing solution may be applied to the sample application zone 14. Insome embodiments, the amplification reagent and wash solution may beadded upstream of the sample application zone.

In some embodiments, the applied sample is allowed to dry. Drying mayinclude activation of a heating element, which is underneath or adjacentto the sample application zone 14. As illustrated in FIG. 1, an optionalheating unit 18 is located near the sample application zone 14. Theheating units may include, but are not limited to, an electrical heater,a chemical heater, an electro-mechanical heater, a radiation basedheat-pad such as IR radiation heat pad. The sample may be dried on thesubstrate to stabilize the sample for a longer period of time.

The sample, which applied to the substrate, may be a biological sample,which is procured from physiological or clinical biological sources thatcomprise nucleic acids. In some embodiments, the sample is nucleic acid,in some other embodiments, the sample is a media from DNA or RNAsynthesis; mixtures of chemically or biochemically synthesized DNA orRNA, wherein the sample is applied directly to the substrate followed byamplification or separation.

The nucleic acid may be extracted from cells using cell-lysis when thesample includes cells or tissue. For example, when the sample iscollected from blood, thin sliced tissue, tissue culture cells,bacterial cells, body fluids such as urine, sputum, semen, secretionscomprising DNA and/or RNA, sample is treated with a lysis reagent afteror before applying it to the substrate. As such, in these embodiments,the method typically further comprises contacting the sample with alysis reagent.

The sample may be pre-treated prior to applying to the sampleapplication zone 14 with an additional lysis reagent for lysing cellswhich are difficult to lyse. For example, cells of Mycobacteriumtuberculosis, which have a complex cell-wall structure that isimpermeable and difficult to lyse, may be pre-treated with a lysisreagent before applying to the substrate.

In some embodiments of the method, the sample itself comprises a lysisreagent. In some other embodiments, the lysis reagent is impregnated inthe sample application zone 14 of the substrate. The cells are lysedwhen contacted with the lysis reagents to extract nucleic acids from thecells. An example of a method for preparing a sample solution comprisingnucleic acids from a biological sample comprises the step of lysing thebiological sample using a lysis reagent, wherein the lysis reagentcomprises chaotropic substances and/or other reagents.

In one or more embodiments, the method further comprises flowing anamplification buffer through the sample application zone along thelength of the substrate for washing the substrate before amplification.The movement of the flow across the length of the substrate is referredto herein as a “lateral flow”.

The term “washing buffer” may interchangeably be used herein as a washbuffer or washing reagent or wash reagent. The washing step may washesaway or dilute the impurities or inhibitors present on the sample ladensubstrate. In some embodiments, the aqueous buffer or amplificationbuffer may dilute the inhibitors present on the substrate. In some otherembodiments, the aqueous buffer or amplification buffer washes away theinhibitors present on the substrate. As noted the term, “washes away”may refer to remove the inhibitors completely or partially from thesubstrate using a buffer, such as aqueous buffer. As noted the term,“dilute”, may refer to reduce the concentration of the inhibitors fromthe substrate using a buffer, such as aqueous buffer. The washing stepmay result in complete or partial removal of the lysis reagents, and/orstabilizing reagents impregnated in the substrate, which may inhibitdownstream applications, such as amplification.

The washing buffer or wash reagent may comprise an aqueous buffer or anamplification buffer without enzymes such as DNA polymerase. The aqueousbuffer may be any solvent of nucleic acids. In some embodiments, theaqueous buffer is at least one of tris(hydroxymethyl)aminomethane andethylenediaminetetraacetic acid (TE) buffer, and PBS or TE in which thetris buffer is substituted with HEPES.

The inhibitors or contaminants may also result from cell lysis, such ascell-debris or other cellular organelle, which have inhibitory effect ondownstream processes and are removed by washing. In some embodiments,the washing dissolves the fuses 16, which subsequently activates theamplification zone heating unit 22. The amplification reagent may flowthrough the length of the substrate upon either dissolving the fuses orupon removal of the fuses.

In some embodiments, the method further comprises flowing a washingbuffer along the substrate. Referring to FIG. 1, the wash reagent may bestored in a wash reagent reservoir 26, and the wash reagent is flowedfrom the reservoir 26 to the wash reagent wicking pad 30 through thesample application zone 14. The wicking force inherent from the porosityof the bibulous substrate, such as a quartz fiber filter itself acts asthe driving force to enable the amplification buffers to flow along thequartz fiber filter and through the sample application zone. The wickingpads 28, 30 draw the amplification and the washing buffers to flowtowards the wicking pad 28, 30 based on its strong wicking force.

In some embodiments, the wash reagent and the amplification buffer arethe same, and may be stored in a single reservoir. In these embodiments,the amplification buffer may comprise amplification reagents except anenzyme, such as polymerase. In these embodiments, the washing solutionmay be replaced by amplification buffer, which may eliminate the step ofwashing during nucleic acid separation by combining the two steps, suchas washing and separation of nucleic acids into one. In theseembodiments, the nucleic acids are washed by diffusion of amplificationbuffer over the substrate 12. The washing buffer or amplificationreagent solution flows along the substrate 12 under the wicking force,wherein no external force is used, and carries away the impuritieshaving a less affinity to the quartz fiber filter than nucleic acids.

The amplification buffer or wash buffer carries away or diluting theimpurities from the sample or substrate with sample because of differentaffinities of impurities and nucleic acids to the substrate. Forexample, a porous quartz fiber filter, thereby eliminating the needs forinstruments to generate the external driving force (e.g. centrifugationforce and pressure) and personnel with specific skills and enablingisolation on site and in remote areas. It appears that by flowing theamplification buffer through the sample application zone to carryimpurities away, the nucleic acids become sufficiently separated fromother components in the sample solution.

The method further comprises flowing a nucleic acid amplificationreagent across a length of the substrate through the sample applicationzone. The terms “amplification reagent” and “amplification reagentsolution” are interchangeably used hereinafter. The amplificationreagent comprises a mixture of dNTP's, oligomer (primer), enzyme(s)including polymerase and amplification buffer.

In some embodiments, the amplification buffer, comprising a mixture ofdNTP's, oligomer (primer), buffer and salts, is added to the substrateto rehydrate the substrate. To start the amplification reaction, theenzyme is added to the substrate separately. In some embodiments, theamplification reaction mixture starts amplification in the presence ofthe amplification buffer when in contact with the target nucleic acidsat the sample application zone, wherein the amplification reactionmixture contains the enzyme. In some embodiments, the amplificationreagents comprising dNTP mixture, oligomers, and amplification bufferreagents may be impregnated in the substrate, which may be reconstitutedusing an aqueous buffer. In these embodiments, the DNA polymerase isadded before starting the amplification reaction. The amplificationreagents may also comprise modified nucleotides.

Referring to FIG. 1, the amplification reagent may be stored in anamplification reagent reservoir 24. On completion of washing, the fuses16 are dissolved and the lateral flow of amplification reagent startsflowing from the amplification reagent reservoir 24 and passes throughthe sample application zone 14 and the detection zone 20 to theamplification reagent wicking pad 28. The wicking pad generates awicking force which enables the lateral flow of amplification reagent tomigrate towards the wicking pad 28, across the length of the substrate.

In one or more embodiments, the amplification reagent flows through thesubstrate to amplify the target nucleic acid to form a nucleic acidamplification product (amplicons). In these embodiments, the targetnucleic acid having a first molecular weight is substantiallyimmobilized at the sample application zone 14 and the amplificationproduct having a second molecular weight migrates away from the sampleapplication zone. The amplification products may migrate via lateralflow. As noted, the terms “first”, “second”, and the like, as usedherein do not denote any order, quantity, or importance, but rather areused to distinguish one element from another.

The nucleic acids having a first molecular weight may be substantiallypositioned around the sample application zone 14. The nucleic acidshaving a second molecular weight are substantially positioned around thedetection zone 20. The term “substantially” used herein refers to aquantity of nucleic acids having certain molecular weights andpositioned around the positioning portion, which may be at least about15% of the total amount of nucleic acids having the molecular weights inthe sample solution retains at the particular position, at least 50% ofthe total amount of nucleic acids having the molecular weights in thesample solution retains at the particular position, or at least 90% ofthe total amount of nucleic acids having the molecular weights in thesample solution retains at the particular position. For example,substantially the nucleic acids with first molecular weight means 90% ofthe total target nucleic acids applied to the substrate retains at thesubstrate at or around the sample application zone 14, and for nucleicacids with second molecular weight, 90% of the total amplified nucleicacids generated on the substrate retains at the substrate at or aroundthe length of the substrate or, specifically, at or around the detectionzone.

In some embodiments of methods, the amplification begins as theamplification reagents enter the sample application zone 14. In someembodiments, the amplification reaction starts when impregnatedamplification reagents are rehydrated to reconstitute the reagents andnucleic acid polymerase added to the substrate. The amplificationreagent may continue to flow through the detection zone 20 to thewicking pad 28. The amplification products may be captured in thedetection zone 20 by one or more capturing agent or probe, such asprimers. In some embodiments, the methods provide a continuous flow ofamplification reagents and the amplification products through thedetection zone. The amplification reagent may move from the reservoir tothe wicking pad via a lateral flow, without forming a bolus.

In some embodiments, the method comprises flowing the nucleic acidamplification reaction mixture which separates the target nucleic acidsand the amplification product according to their molecular weights. Thetarget nucleic acids have first molecular weight and the amplifiednucleic acids have second molecular weight, wherein the differencebetween the molecular weight may enable the amplified nucleic acids tobe separated from the target nucleic acids during lateral flow. As canbe seen from the examples, after diffusion of the amplification reagentsthrough the sample application zone 14 along the length of the substrate12, nucleic acids are positioned on the quartz fiber filter 12 accordingto the molecular weights thereof. To be specific, nucleic acids havinghigher molecular weights are positioned closer to the sample applicationzone 14 than nucleic acids having lower molecular weights.

The sample application zone 14 is the positioning portion for thenucleic acids having the first molecular weight. A substantial portionof the nucleic acids having a first molecular weight are positionedaround the sample application zone 14. In some embodiments, the firstmolecular weight is in a range of at least about 50 kb. In someembodiments, the first molecular weight is in a range from about 50 kbto about 150 kb. In some embodiments, about 50 kb refers to a range of50 kb±15 kb.

In some embodiments, substantial portion of the nucleic acids having asecond molecular weight are positioned around the second end 31. In thiscase, the second end 31 is the positioning portion for the nucleic acidshaving the second molecular weight. The second molecular weight nucleicacids may be distributed across the substrate. In some embodiments, thesecond molecular weight is in a range of less than about 50 kb. In someembodiments, the amplicons may have more than one molecular weightpopulation, which may results from more than one target molecules.

In some embodiments of the method, one or more amplification reactionsoccur on the substrate. In some embodiments, a first amplificationreaction occurs at the sample application zone to generate a firstamplification product. One or more amplification reactions may occurduring migration of the first amplification product. Similarly, one ormore amplification reactions may occur during migration of the secondamplification product, and so on. Multiple amplification reactionsgenerate plurality of amplification products, which facilitatesdetection method with greater ease, sensitivity and accuracy.Especially, the multiple amplification reactions are useful when thetarget nucleic acid is available in a trace quantity, for example,sample procured for forensic application or from biopsy sample.

In some embodiments, the amplification is an isothermal amplificationreaction. The isothermal amplification may include but is not limitedto, rolling circle amplification (RCA), multiple displacementamplification (MDA), helicase dependent amplification (HDA), ping pongamplification, cross priming amplification (CPA), recombinase polymeraseamplification (RPA), loop mediated isothermal amplification (LAMP) andstrand displacement amplification (SDA).

The amplified nucleic acids or amplicons may be detected on thesubstrate. In some embodiments, either the substrate comprises detectionprobes or the detection probes may separately be added during, prior oron completion of the amplification reaction. In some embodiments, asolution comprising one or more detection probes is added to thesubstrate. In some other embodiments, the detection probes are part ofthe amplification reaction mixture. A solution comprising one or moredetection probes or an amplification reaction mixture comprising one ormore detection probes may be flowed along the length of the substrate.In some embodiments, the detection probes may directly be added to thedetection zone 20. In some examples, the detection probes in a form ofsolution or as a part of an amplification reaction mixture is applied atthe detection zone 20.

The capturing probes may capture the amplified nucleic acids of intereston the substrate during diffusion of the probes. The term “capture” mayinclude but are not limited to hybridization of the amplified nucleicacids with the probes, physical interaction of the probes with theamplified nucleic acids, or chemical interaction of the probes with theamplified nucleic acids. The amplification product may be immobilized onthe substrate by a physical interaction with the substrate, using acapturing probe, using a detection probe or combinations thereof. Insome embodiments, the amplification product is captured on the substrateby binding with a capturing probe physically bound to the substrate toform captured amplification product. The nucleic acids may be capturedby the capture probe by hybridization, for example, when the captureprobe is a primer. The captured amplification product may further bindto a detection probe for detection of amplicons.

The “detection probe” may detect the amplicons using one or moredetection method. The detection probes may include, but are not limitedto, antisense oligomer, pyrophosphate, phosphatase, biotin-streptavidinbeads, antibody, fluorescence resonance energy transfer (FRET) probes,horseradish peroxidase (HRP) probes and luciferase. The antisenseoligomers may comprise of natural nucleotides or nucleotide analogs. Theoligonucleotides may be labeled with FRET probes, such as fluorescein,Cy5, Cy5.5, and BIODPY®.

The separated nucleic acids may be detected by various procedures. Insome embodiments, the nucleic acids such as DNA may be detected bysouthern blot and RNA may be detected by northern blot. The nucleicacids are separated at the detection zone, may be detected bycolorimetric detection method, chemical, electrical, pH, luminescent orfluorescence based detection method.

In some embodiments, the detection probe is an antisense oligomer whichhybridizes with the amplified nucleic acids, wherein the antisenseoligomer probe is attached to a molecular marker which can be detected.The molecular marker may include but is not limited to, radioactivemolecules, fluorescent molecules, proteins or peptides. For example, aradioactive isotope of phosphorus ³²P is inserted in the phosphodiesterlinkage of the antisense oligomer, which may function as a detectionprobe. The detection probe may be tagged with a non-radioactive marker,such as digoxygenin. In this case, anti-digoxygenin antibody may be usedto detect the digoxygenin labelled probe. In some examples, thedetection probe is a chemical entity, which in contact with a moietyattached to the amplified nucleic acids may generate fluorescencesignal. The detection probe may be an enzyme, which on interaction witha moiety on the amplified nucleic acids may produce a chemical whichgenerates a color. This may distinguish the colored amplified productfrom the colorless target nucleic acids.

In some embodiments, the method further comprises: flowing a solutioncomprising primary detection probes through the sample application zonealong the length of the substrate to bind to the captured amplificationproduct to form a primary detection probe bound amplification product.Some embodiments of the method further comprises: flowing a solutioncomprising secondary detection probes through the sample applicationzone along the length of the substrate to bind to the primary detectionprobe bound amplification product. In some embodiments, the primarydetection probe may be attached to a fluorescence moiety, wherein thesecondary detection probe may be selected as a quencher. The secondarydetection probe may quench the fluorescence generated by the primarydetection probe on interaction. In some other embodiments, the primarydetection probe may be attached to a primary antibody, wherein thesecondary detection probe may be selected as a secondary antibody,wherein the secondary antibody may bind to the primary antibody andgenerate a signal.

In some embodiments, after extraction and separation of the nucleicacids, such as DNA and/or RNA, the nucleic acids may be stabilized forextended storage, depending on its application and requirement. Theamplification product may be physically bound to the substrate, whereinthe binding efficiency may be further increased using various reagents.In case of stabilization, the stabilizing reagents may be impregnated inthe substrate. In some embodiments, the stabilizing reagents may beimpregnated at the sample application zone 14, sample detection zone 20or in the entire substrate 12. The stabilization reagents are describedin more detail in later part of the specification.

In some embodiments, the method further comprises applying a flowbarrier to the substrate to immobilize the amplification products byobstructing the amplification product flow. The fuses 16 may also bereferred to herein as flow barriers. The amplification product may notflow further until the barrier is removed. The flow barrier may be atransverse section, disposed on the longitudinal strip of the substrate12. The shape of the flow barrier may be any regular shape such asrectangular, square planar, spherical shape or any irregular shape. Theflow barrier may be made of polymer, glass, wood, metal or combinationsthereof. In some embodiments, the flow barrier comprises quartz, paper,sugar, salts or combinations thereof. In some embodiments, the flowbarrier is located adjacent to the substrate 12 and after diffusion ofthe amplification buffer through the sample application zone 14 alongthe length of the substrate 12, a substantial portion of the nucleicacids are positioned at the interface between the sample applicationzone 14 and the flow bather. In some embodiments, the flow bather 16 isan elongated strip made of a material other than quartz fiber filter. Insome embodiments, the flow barrier is made of cellulose, such ascommercially available cellulose, for example, 31ETF (Whatman®).

In one or more embodiments of the method, a migration modifier is addedto the substrate. In some embodiments, the sample application zonefurther comprises a migration modifier. The migration modifier may beused to modify the migration rate or pattern of the amplificationproduct through lateral flow. The migration modifier may decrease themigration rate of one or more target molecules by ensuring betterbinding of the target nucleic acids to the substrate. The migrationmodifier modifies binding efficiency of the molecules to the substrate.Use of migration modifier ensures efficient separation and detection ofthe amplified product.

In some embodiments, the migration modifier comprises a chaotrope. Themigration modifier may comprise a guanidinium salt, which may minimizethe migration of the target DNA during the lateral flow of the amplifiedproduct DNA. In one embodiment, the migration modifier comprisesguanidinium thiocyanate. Generally, guanidinium thiocyanate improvesbinding of genomic DNA to the substrate, which retains the targetnucleic acid, such as genomic DNA bound at the sample application zone.

In some embodiments, the method further comprises providing a wickingpad adjacent to the second end 31 of the substrate, which may functionas a stopping pad or collection pad. The stopping pad may stop the flowof amplified nucleic acids near the second end. The stopping pad may besubstituted by a flow bather. The collection pad may collect the nucleicacids from the substrate by transferring the amplicons to the collectionpad.

In some embodiments, the method further comprises adding a collectionpad. In these embodiments, after diffusion of the washing buffer throughthe sample application zone 14 along the length of quartz fiber filter12, the collection pad (a stopping pad, a wicking pad, or a quartz fiberfilter) is disconnected from the substrate and replaced with a newcollection pad so that the amplification buffer flows from the substrateto the new collection pad.

The amplification reagents or amplification buffer flows along thelength of the quartz fiber filter and through the sample applicationzone to migrate nucleic acids on the quartz fiber filter, the lower themolecular weight, the further nucleic acids migrate on the quartz fiberfilter from the sample application zone. When the amplification or washbuffer flows from the quartz fiber filter to a stopping or wicking padmade of a material other than the quartz fiber filter, nucleic acidsmigrating with the aqueous buffer will stop and be positioned at aninterface between the quartz fiber filter and the stopping or wickingpad.

One or more embodiments of a substrate comprise a sample applicationzone for applying a sample comprising a target nucleic acid having afirst molecular weight and flowing an amplification reaction mixturealong the length of the substrate through the sample application zoneforming a nucleic acid amplification product having a second molecularweight. In some embodiments, the substrate further comprises a detectionzone for separating the nucleic acid amplification product having secondmolecular weight from the target nucleic acids having first molecularweight according to molecular weights of the amplified nucleic acids andtarget nucleic acids.

In some embodiments, the substrate is a solid substrate, which is anon-water dissolvable material, which enables collection, extraction,separation, detection, storage of nucleic acids and combinationsthereof, followed by elution without solubilizing the material usingwater or aqueous buffer.

In some embodiments, the substrate is an elongated strip comprising afirst end 11, a sample application zone 14, and a second end 31. The runtime starting from sample application to separation may increase withincreasing the length of the substrate, however the separation of thenucleic acids are better with increasing the length of the substrate.The length of the substrate may be optimized considering betterseparation as well as run time. The substrate may have a length in arange between 1 cm and 20 cm. In some embodiments, the substrate has alength less than 10 cm.

The substrate includes, but is not limited to, materials such ascellulose, cellulose acetate, nitrocellulose, glass fibers orcombinations thereof. In one embodiment, the substrate comprisescellulose. In one or more embodiments, the substrate is selected from anitrocellulose membrane, a cellulose membrane, a cellulose acetatemembrane, a regenerated cellulose membrane, a nitrocellulose mixed estermembranes, a polyethersulfone membrane, a nylon membrane, a polyolefinmembrane, a polyester membrane, a polycarbonate membrane, apolypropylene membrane, a polyvinylidene difluoride membrane, apolyethylene membrane, a polystyrene membrane, a polyurethane membrane,a polyphenylene oxide membrane, apoly(tetrafluoroethylene-co-hexafluoropropylene) membrane, and anycombination of two or more of the above membranes. In some embodiments,the substrate comprises modified cellulose, such as pegylated celluloseor pegylated nitro cellulose.

In some embodiments, the substrate is a porous substrate. In oneembodiment, the substrate is a porous cellulose membrane. In oneembodiment, the solid substrate is a porous cellulose paper, such as acellulose substrate from GE Healthcare Life Sciences (formerlyWhatman™). In one example, the cellulose substrate comprises903-cellulose, FTA™ or FTA™ Elute.

The substrate may be any porous and bibulous filter to which the samplesolution comprising nucleic acids may be sorbed and which does notinhibit storage or subsequent analysis of the nucleic acid appliedthereto. In some embodiments, the substrate is a quartz fiber filter. Insome embodiments, the quartz fiber filter is made of pure quartz fiberswith no binders. In some embodiments, the quartz fiber filter has aparticle retention efficiency of about 98% for particles of a size of noless than 2.2 μm, a basis weight of 85 g/m², and a thickness in a rangeof from about 300 μm to about 600 μm. Examples of quartz fiber filterssuitable for this purpose include, but are not limited to, Whatman®grade QM-A quartz microfiber filters available from GE HealthcareBio-Sciences Corp., New Jersey, USA), and AQFA quartz fiber filtersavailable from Millipore Corporation, Billerica, Mass., USA.

The sample solution comprising nucleic acids is applied to the sampleapplication zone 14 of the quartz fiber filter 12. The sampleapplication zone 14 may be in any shape or configuration that the samplesolution may be applied thereto. In some embodiments, the sampleapplication zone 14 of the quartz fiber filter 12 comprises a lysisreagent and the biological sample comprising nucleic acids is directlyapplied to the sample application zone 14 of the quartz fiber filter 12.In one or more embodiments of the device, the sample application zonecomprises an FTA pad.

In one or more embodiments, the substrate comprises one or morecell-lysis reagents, protein denaturing agents or stabilizing agents ina substantially dry state. In other embodiments, the substrate furthercomprises buffer reagents, reducing agents, and optionally free-radicalscavengers in addition to protein denaturing agents in a dry state. Thesubstrate may extract nucleic acids and preserve nucleic acids under dryconditions, wherein the dried nucleic acids may further be eluted fromthe substrate by re-hydrating with water or aqueous buffer.

As noted, the sample application zone comprises a lysis reagent, whereinthe lysis reagent may comprise chaotropes. The examples of chaotropicsubstances include, but are not limited to, guanidinium hydrochloride,guanidinium chloride, guanidinium isothiocyanate/thiocyanate, sodiumthiocyanate, sodium perchlorate, sodium iodide, potassium iodide, urea,and/or any combination thereof. A typical anionic chaotropic series,shown in order of decreasing chaotropic strength, includes: CCl₃COO⁻,CNS⁻, CF₃COO⁻, ClO₄ ⁻, I⁻, CH₃COO⁻, Br⁻, Cl⁻, or CHO₂ ⁻. The lysisreagent may include chaotropic substances in concentrations of from 0.1M to 10 M, or from 1 M to 10 M.

For some of the biological samples, such as bacteria, the lysis reagentmay comprise, for example, lytic enzymes or the biological samples maybe pretreated, for example, with lytic enzymes, prior to being lysed.

In some embodiments, the lysis reagent also includes a sufficient amountof buffer. The examples of buffers for use in the lysis reagent includetris-(hydroxymethyl) aminomethane hydrochloride (Tris-HCl), sodiumphosphate, sodium acetate, sodium tetraborate-boric acid andglycine-sodium hydroxide.

In some embodiments, the lysis reagent also includes a non-ionicsurfactant, a cationic surfactant, an anionic surfactant, an amphotericsurfactant, and/or any combination thereof. Exemplary nonionicsurfactants include, but are not limited to,t-octylphenoxypolyethoxyethanol (TRITON X-100™),(octylphenoxy)polyethoxyethanol (IGEPAL™ CA-630/NP-40),triethyleneglycol monolauryl ether (BRIJ™ 30), sorbitari monolaurate(SPAN™ 20), or the polysorbate family of chemicals, such as polysorbate20 (i.e., TWEEN™ 20), TWEEN™ 40, TWEEN™ 60 and TWEEN™ 80 (Sigma-Aldrich,St. Louis, Mo.). Examples of cationic surfactants includecetyltrimethylammonium bromide, dodecyltrimethylammonium chloride,tetradecyltrimethylammonium chloride and cetylpyridinium chloride.

The concentration of the surfactant in the lysis reagent could varyslightly among the different surfactants and depending on the componentsin the biological sample to be lysed. In some embodiments, theconcentration of the surfactant is in a range of from about 0.01% toabout 20% by weight. The lysis reagent may further comprisedithiothreitol (DTT).

The lysis reagent may also comprise protease, such as serine, cystineand metallic proteases. A protease free of nuclease may be used. Aprotease comprising a stabilizer, such as metallic ions, may be used.The protease may be used, upon addition, in an amount of preferably fromabout 0.001 IU to about 10 IU, more preferably from about 0.01 IU toabout 1 IU, per ml of the whole lysis reagent.

The lysis reagent may comprise a defoaming agent. Examples of thedefoaming agent include silicon-comprising defoaming agents (e.g.,silicone oil, dimethylpolysiloxane, silicone emulsion, modifiedpolysiloxane and silicone compound), alcohol series defoaming agents(e.g., acetylene glycol, heptanol, ethylhexanol, higher alcohol andpolyoxyalkylene glycol), ether series defoaming agents (e.g., heptylcellosolve and nonyl cellosolve-3-heptylsorbitol), fat-and-oil seriesdefoaming agents (e.g., animal oils and plant oils), fatty acid seriesdefoaming agents (e.g., stearic acid, oleic acid and palmitic acid),metallic soap series defoaming agents (e.g., aluminum stearate andcalcium stearate), fatty acid ester series defoaming agents (e.g.,natural wax and tributyl phosphate), phosphate series defoaming agents(e.g., sodium octylphosphate), amine series defoaming agents (e.g.,diamylamine), amide series defoaming agents (e.g., stearic acid amide),other defoaming agents (e.g., ferric sulfate and bauxite), and anycombination thereof.

The lysis reagent may comprise an alcohol. Any of a primary alcohol, asecondary alcohol and a tertiary alcohol may be used. Methyl alcohol,ethyl alcohol, propyl alcohol, butyl alcohol, and an isomer thereof maypreferably be used. The concentration of alcohol in the lysis reagent ispreferably in a range of from about 5% to about 90% by weight.

In some embodiments, the lysis reagent is RA1 lysis buffer from IllustraRNAspin Mini kit (cat#25-0500-71) supplemented with 0.35 μl2-beta-mercaptoethanol (BME, cat#60-24-2).

The method for preparing the sample solution comprising nucleic acidsmay be conducted with the aid of, for example, a ultrasonic wavetreatment, a treatment using a sharp projection, or a high-speedstirring or vortexing treatment.

In one embodiment, the substrate is impregnated with nucleic acidstabilizing reagents. These stabilizing reagents may includeDNA-decomposing enzyme inhibitor, such as DNAse inhibitor and/orRNA-decomposing enzyme inhibitor, such as RNAse inhibitor, bufferreagents, or chelating agents (e.g., EDTA).

As noted, the substrate comprises an RNase inhibitor, wherein the RNaseinhibitor comprises vanadyl ribonucleoside complex (VRC), a nucleotideanalogue, a commercially available RNase inhibitor (e.g., SUPERase-In™),or a triphosphate salts, such as sodium triphosphate.

The substrate may comprise DNAse inhibitor, which may include but is notlimited to, 2-mercaptoethanol, 2-nitro-5-thiocyanobenzoic acid, Actin,Alfatoxin B2a, G2, G2a, and M1, Ca²⁺, EGTA, EDTA, Sodium dodecylsulfate, Calf spleen inhibitor protein, Carbodiimide and cholesterolsulfate, Iodoacetate.

In some embodiments, the substrate comprises stabilizing reagent, whichmay include a reducing agent that facilitates denaturation of RNase andaids in the isolation of undegraded RNA. Exemplary reducing agentincludes, but is not limited to, 2-aminoethanethiol,tris-carboxyethylphosphine (TCEP), and β-mercaptoethanol.

As noted, the substrate further comprises a chelating agent, wherein thechelating agent is selected from ethylenediaminetetraacetic acid (EDTA),citric acid, ethylene glycol tetraacetic acid (EGTA) or combinationsthereof.

The substrate may further comprise a UV protectant, a free-radicalscavenger, a chelator or combinations thereof for stabilizing nucleicacids. Without intending to be limited to any specific UV protect, anexemplary antioxidants include, for example, hydroquinone monomethylether (MEHQ), hydroquinone (HQ), toluhydroquinone (THQ), uric acid, andascorbic acid. In some embodiments, the antioxidant is THQ.

As noted, in some embodiments, the substrate comprises one or moredetection probes. The detection probes may be located at the sampleapplication zone, downstream of the sample application zone orcombinations thereof. In one embodiment, one or more detection probesare located on the substrate 12, downstream 20 of the sample applicationzone 14.

The detection probes may be impregnated in the substrate. In someembodiments, the detection probes are impregnated in the substrate underdried condition, wherein the impregnated detection probes may berehydrated during the process of amplification or after theamplification reaction is over. The impregnated detection probes may bereconstituted and activated by rehydration.

In one embodiment, the device comprises a substrate and an amplificationreagent reservoir. In some embodiments, the device further comprises awash reagent reservoir.

Referring to FIG. 1, the device 10 comprises a substrate 12 having afirst end 11 and a second end 31. In some embodiments, the substrate isan elongated strip. The device 10 further comprises an amplificationreagent reservoir 24, a wash reagent reservoir 26, an amplificationreagent wicking pad 28 and a wash reagent wicking pad 30. In someembodiments, the substrate 12 either directly or indirectly coupled tothe amplification reagent reservoir 24, wash reagent reservoir 26, andthe wicking pads 28 and 30. In an embodiment of the device, theamplification reagent reservoir 24, sample application zone 14 andamplification reagent wicking pad 28 are on a first straight line, andthe wash reagent reservoir 26 and the wash reagent wicking pad 30 arepresent on a second straight line which is perpendicular to the firststraight line. Any other arrangement that maintains similar connectivityof different components of the device may also be possible. Although theterm “coupled” refers to connected and often is used to describephysical or mechanical connections or couplings, the term is notintended to be so restricted and can include direct or indirectconnections or couplings.

The substrate 12 comprises a sample application zone 14. The sampleapplication zone 14 may also be used as a sample lysis zone and/ornucleic acid stabilization zone. The substrate further comprises fuses16, which retain the sample at the sample application zone, and do notallow the sample to flow outside of the boundary formed by the fuses 16.The substrate further comprises a detection zone 20, which comprises acontrol line and a test line. The detection zone 20 is located at theopposite end of the sample application zone 14 of the longitudinal strip12. In one or more embodiments, the substrate further comprises anamplification zone heating unit 22 covering the whole longitudinalstrip. The amplification zone heating unit may require maintaining anisothermal condition for amplification reaction. In some embodiments,the substrate optionally contains a heating unit for drying the loadedsample 18. In some embodiments, the substrate 12 is made of quartz fiberfilter.

In one embodiment, a device 10 comprises an elongated strip of quartzfiber filter 12. The quartz fiber filter 12 includes a first endcomprising a sample application zone 14 and a detection zone 20 at asecond end opposite to the first end 14.

The device comprises one or more wicking pads. The wicking pad may be aporous matrix. In some embodiments, the porous matrix may be bibulous,to which the sample solution comprising nucleic acids is sorbedefficiently. The bibulous porous matrix does not inhibit storage orsubsequent analysis of the nucleic acid applied thereto. In someembodiments, the wicking pad is a quartz fiber filter. The quartz fiberfilter may be made of pure quartz fibers with no binders. In someembodiments, the quartz fiber filter has a particle retention efficiencyof about 98% for particles of a size of no less than 2.2 μm, a basisweight of 85 g/m², and a thickness in a range of from about 300 μm toabout 600 μm. Examples of quartz fiber filters suitable for this purposeinclude, but are not limited to, Whatman® grade QM-A quartz microfiberfilters available from GE Healthcare Bio-Sciences Corp., (New Jersey,USA), and AQFA quartz fiber filters available from Millipore Corporation(Billerica, Mass., USA). The wicking pad may be a strip of bibulousmaterial, such as cellulose, silica microfiber filter or glass fiber. Insome embodiments, the wicking pad 28, 30 is Whatman® grade 470 specialpurpose filter papers commercially available from GE Healthcare, NewJersey, USA.

FIG. 2 illustrates another embodiment of a device 32, wherein the deviceis similar to the device described in FIG. 1, except the fact that, theamplification reagent reservoir 24 and the wash reagent reservoir 26 arecoupled to each other. In some embodiments, the amplification reagentreservoir 24 and the wash reagent reservoir 26 are adjacent to eachother, configured such that the wash reagent reservoir is directlycoupled to the substrate 12. The amplification reagent reservoir isindirectly coupled to the substrate 12, through the wash reagentreservoir 26. In this embodiment, both the amplification reagentreservoir 24 and the wash reagent reservoir 26 are aligned on a straightline with the sample application zone 14 and the wash reagent wickingpad 30. This configuration results in supplying the wash reagent firstto the substrate 12, through the sample application zone 14 andextracted out the impurities or inhibitors, if present at the sampleapplication zone, to the wicking pad 30. On completion of washing, theamplification reagents stored in the reservoir 24 starts migrating tothe substrate 12 through the wash reagent reservoir 26. Theamplification reaction starts when the amplification reagent comes incontact with the target nucleic acids at the sample application zone 14.The device comprises a heating unit 22 for amplification zone, whichhelps in maintaining a constant temperature for substrate duringamplification reaction. The device may further comprise a sample heatingunit 18 for drying the nucleic acids for stabilization.

FIG. 3 illustrates another embodiment of a device 34, wherein the deviceis similar to the device described in FIG. 1, except the fact that, thedevice further comprises a wicking pad 36 near the sample applicationzone 14. The fuses 16 are disposed such that they form a boundarystarting from sample application zone 14 and ending at wicking pad 36.In an embodiment of the device, the amplification reagent reservoir 24,sample application zone 14 and amplification reagent wicking pad 28 areon a first straight line, and the wash reagent reservoir 26 and the washreagent wicking pad 30 are present on a second straight line which isperpendicular to the first straight line. Any other arrangement thatmaintain similar connectivity of different components of the device mayalso be possible. In this embodiment, the amplification reagentreservoir 24, the wash reagent reservoir 26, and the wicking pads 28 and30 are directly coupled to the substrate 12. The wash reagent is firstsupplied to the substrate 12, through the sample application zone 14 andextracted out the impurities or inhibitors, if present at the sampleapplication zone, to the wicking pad 30. On completion of washing, theamplification reagents stored in the reservoir 24 starts migrating tothe substrate 12. The amplification reaction starts when theamplification reagent comes in contact with the target nucleic acids atthe sample application zone 14. The device of this embodiment alsocomprises a heating unit 22 for amplification zone and a sample heatingunit 18.

Referring to FIG. 4, which illustrates another embodiment of a device32. The device of this embodiment is similar to the device described inFIG. 2, wherein the device of FIG. 4 comprises only one reservoir, suchas amplification reagent reservoir 24 which comprises the amplificationreagent, which is directly coupled to the substrate 12. The device doesnot specifically need to contain any washing reagent or a washingreagent reservoir. In these embodiments, the amplification reagent mayfunction as the wash reagent. In this embodiment, the amplificationreagent reservoir 24 is aligned on a straight line with the sampleapplication zone 14 and the wash reagent wicking pad 30. In thisembodiment, the amplification reagent flows to the substrate 12, throughthe sample application zone 14 and extracts out the impurities orinhibitors, if present at the sample application zone, and to thewicking pad 30. On completion of washing, the amplification reagentsstored in the reservoir 24 further migrate to the substrate 12. Theamplification reaction starts when the amplification reagent, comprisingthe enzyme, comes in contact with the target nucleic acids at the sampleapplication zone 14. The amplification reagent serves as the washreagent until the lysis reagent in the sample application pad (e.g.,guanidinium thiocyanate) is sufficiently removed. The lysis reagentinhibits amplification, so once inhibitors are removed, theamplification reagent including the polymerase starts amplifying thenucleic acids. The device comprises a heating unit 22 for amplificationzone, which helps in maintaining a constant temperature for substrateduring amplification reaction. The device may further comprise a sampleheating unit 18, for drying the nucleic acids for stabilization. In someembodiments, the heating unit supplies heat necessary for cell lysis.

In addition to in-solution and in-paper lysis approaches, two classes oftopologies may be used for amplification, such as (1) amplificationduring lateral flow or amplification-in-flow 42, 46, as shown in FIGS.5A and 5C; and (2) amplification restricted in a defined place oramplification-in-place 44, 48, as shown in FIGS. 5B and 5D.Amplification-in-flow requires that the target nucleic acids, such asDNA have high molecular weight, wherein the amplification is initiatedin the sample application zone. The low molecular weight amplicons thatare generated may migrate downstream of the substrate, wherein thatportion of the substrate has an amplification zone heating unit 22 andthe amplicons continue to amplify further, yielding a semi-continuousdelivery of amplicons to the detection line 20.

Alternatively, amplification in-place topologies use fuses, such asvalves, gates, switches or fuses in the flow path to ensure thatamplification occurs in a largely static fluid volume within theamplification zone comprising a heating unit 22. The amplicons areallowed to flow on to the detection zone only after the desiredamplification time has elapsed by opening or closing of a valve or lapseof a delay circuit 16.

Some other embodiments of a device are illustrated in FIGS. 5A, 5B, 5Cand 5D, wherein the device illustrates in flow and in-place topologywhich comprise an amplification reagent reservoir 24, a substrate 12 anda wicking pad 28. In an embodiment, the amplification reagent reservoir24, the substrate 12 and the wicking pad 28 are located in a straightline. The substrate comprises a sample application zone 14, a detectionzone 20 and a heating unit for amplification 22. The detection zone 20comprises a test line. The test line is where the amplicons are capturedfor detection, for example, using colored beads hybridized to thecaptured amplicons for detection. The devices of FIGS. 5B, 5C and 5Dadditionally have one or more fuses 16. In the presence of a fuse, theamplification reaction mixture does not flow through the substrate. Inthe absence of fuse, the amplification reaction mixture flows throughthe substrate 12, followed by amplifying the target nucleic acids andseparating the nucleic acids based on molecular weight in the detectionzone 20.

The quartz fiber filter, the collection pad, the wicking pad, thestopping pad may be supported on a backing material such as a plasticmaterial like polyester (Mylar®) or polyethylene terephthalate (PET).

In some embodiments, the device 10, 32, 34, 40 or 42 comprises a plastichousing (not shown) enclosing the quartz fiber filter 12, the collectionpad or amplification reagent wicking pad 28, the wicking pad 28, 30 thestopping pad therein but exposing the sample application zone 14 withcorresponding opening thereof so that the sample solution comprisingnucleic acids, the washing buffer, and the aqueous buffer may be appliedto the sample application zone 14. In some embodiments, the sampleapplication zone 14 may be located outside of the housing.

The device 10 may be a simple nucleic acidpurification/isolation/separation device, or alternatively may be a partor components of a larger nucleic acid analysis device or system.

As noted, the residual lysis reagents, inhibitors or other impuritiesfrom the sample application zone 14 may be washed off beforeamplification and separation using the amplification buffer. In someembodiments, an additional washing solution may be used, wherein thewashing solution comprises an aqueous buffer, which may be any solventof nucleic acids. In some embodiments, the aqueous buffer comprisestris(hydroxymethyl)aminomethane, ethylenediaminetetraacetic acid (EDTA)buffer, phosphate buffered saline (PBS) or Tris EDTA (TE) in which thetris buffer is substituted with HEPES. As noted, the aqueous bufferflows through the sample application zone 14 along the length of quartzfiber filter 12. In some embodiments, the first end 11 of the substrateis placed into the aqueous buffer so that the aqueous buffer flows fromthe first end 11 of the substrate 12.

The washing buffer may comprise an enzyme capable of degrading acontaminant, e.g., protein. Moreover, it may comprise deoxyribonuclease,ribonuclease or the like depending on circumstances. Use of washingbuffer comprising deoxyribonuclease allows selective recovery of RNA.Similarly, use of a ribonuclease-comprising washing buffer allowsselective recovery of DNA.

The amplification buffer may comprise a water-soluble organic solventand/or water-soluble salt. The washing buffer washes out an impurity ina sample solution, which is adsorbed on the quartz fiber filter togetherwith nucleic acids. Therefore, it may have a composition which desorbsthe impurity from the quartz fiber filter while keeping nucleic acidsadsorbed. A water-soluble organic solvent, e.g., alcohol, in whichnucleic acids are sparingly soluble, is suitable for desorbingcomponents other than nucleic acid from the quartz fiber filter. At thesame time, incorporation of a water-soluble salt enhances the effect ofadsorbing nucleic acids to improve selective desorption of anunnecessary component.

The water-soluble organic solvents may be used as a washing bufferinclude, but are not limited to, methanol, ethanol, isopropanol,n-propanol, butanol and acetone. The water-soluble organic solvent isincorporated in a washing buffer preferably at about 20% to about 100%by weight, more preferably about 40% to about 100% by weight. In oneembodiment, an exemplary water-soluble salt to be included in a washingbuffer is preferably a halide salt or tris(hydroxymethyl)aminomethane.

The amplification buffer for washing the substrate may be applied to thequartz fiber filter at the same place as where the sample solutioncomprising nucleic acids is applied, i.e., the sample application zone.The amplification buffer may also be applied to the quartz fiber filterat a place different from both the sample application zone and thebuffer loading portion.

In some embodiments, the amplification buffer flows to the second end 31(FIG. 6) and carries unwanted contaminants in the sample solution to thesecond end. The second end 31 is then cut off before flowing theamplification reagents or amplification buffer. In such way, the nucleicacids positioned on the remaining quartz fiber filter arepurified/separated/isolated from the unwanted contaminants.

After diffusion of the amplification buffer, the nucleic acidspositioned on the quartz fiber filter may be eluted under conditions oflow ionic strength or with water, respectively.

As used herein, the term “sorb” means that the sample solution isabsorbed, adsorbed or otherwise incorporated into or onto the sampleapplication zone in such a way as not to be readily removed from thesample application zone unless subjected to conditions which areintentionally or inadvertently performed to remove the sorbedcomposition from the sample application zone.

The following examples are included to provide additional guidance tothose of ordinary skill in the art in practicing the claimed invention.These examples do not limit the invention as defined in the appendedclaims.

EXAMPLES Example 1 Preparation of GF/F Substrate

A GF/F porous matrix (Whatman®-GE Healthcare) was soaked in a solutionof 280 mg/ml guanidinium thiocyanate (Sigma-Aldrich) and allowed to airdry. This treated matrix was then cut into 5×5 mm squares and eachsquare assembled on a modified lateral flow strip using PDMS glue. Eachlateral flow strip (substrate) 12 was modified by removing an areaapproximately 10-12 mm from the pointed tip 11 underneath where the 5 mmsquare of treated GF/F porous matrix 14 would reside, as shown in FIG.6. One strip was processed with a wick 28 present on the end oppositethe point 11 (FIG. 8), while the other strip was processed without awick. The GF/F glass fiber comprised elute-chemistry that was bridging agap in a nitrocellulose strip (as shown in FIGS. 6 and 7 A).

Example 2 Preparation of a Modified Porous Nitrocellulose BasedSubstrate

A modified porous matrix was prepared by soaking a nitrocellulose basedsubstrate (GE Healthcare) in an aqueous solution containing 10% (w/v)polyethylene glycol methyl ether methacrylate 300 (PEG; Sigma-Aldrich)and 30% (v/v) Tween 20 (Sigma-Aldrich) for 10 seconds. Excess solutionwas removed and the treated matrix subjected to E-beam (AdvancedElectron Beam) treatment for a total dose of 10 kGy. Followingirradiation, the modified matrix was treated as follows: 1) washed bysoaking three times for 30 minutes each in distilled water using anorbital rotating platform, 2) the excess water removed and 3) allowed toair dry at room temperature overnight.

Example 3 Cell Lysis Using Modified Substrate

The FTA® papers (GE Healthcare) were used to accelerate thermal lysis ofa Staphylococcus test strain. Here, in addition to traditional cellulosebased papers, FTA® chemistry was applied to glass fiber membranes (GF/Fand Standard 17). A summary of the efficacy of these materialsdemonstrating lysis at different temperature and time is shown inTable 1. Furthermore, to determine the effects of incomplete water lossduring sample lysis, each FTA sample was placed in “sealed” (Kaptontape), “open,” or partially-sealed “capped”, (e.g. under a cap with asmall headspace) configurations. As shown in Table 1, successful lysis(S. chromogenes) was achieved for all tested configurations (as shown bycross in Table 1), including sealed and capped configurations,indicating that complete drying is not a requisite for cell lysis.Generally, lower incubation temperatures require longer incubation timeto ensure six-log reduction in viable bacterial load (S. chromogenes),even at 49° C. for isothermal DNA amplification. A complete lysis wasachieved in 10 minutes under the above conditions with FTA Elutechemistry. Similar findings were observed in experiments with S. aureus,and thus 49° C. and 15 minutes (or less) were down-selected for initialintegrated device demonstration to showcase the potential utility ofFTA® solid substrate lysis.

TABLE 1 Efficacy of different materials demonstrating lysis at differenttemperature and time Time (min) at 90° C. Time (min) at 49° C. SubstrateMode 1 2 5 10 10 15 20 FTA Elute Open X X (Cellulose) Capped X X X X X XX Sealed X X X X X X Glass Open X X Fiber + Capped X X X X X EluteSealed X X X

Example 4 Substrate Selection for Amplification

To evaluate FTA® Elute chemistry compatibility with amplification,punches (5 mm×5 mm) were placed on a nitrocellulose (NC) strip with agap (to ensure flow through the paper) and washed with various volumes,as shown in FIG. 7 A. After the fluid in the well was exhausted, thepunch was removed from the strip to prevent backflow during drying. Thepunches were dried in air overnight and then characterized by FTIR-ATRspectroscopy (FIG. 7 B) to determine the degree of washing relative toan unwashed FTA® paper and to a sample of the base paper from which FTA®paper is produced.

Generally the expectation was that, the amplification would not beinhibited if the residual chemistry was reduced to <1% of its initiallevel. The FTIR-ATR data shown in FIG. 7 B, which illustrates that FTA®Elute, in cellulose and glass fiber, were both washed and reduced to <1%residual composition for 25-50 μl (2-5 paper volumes) depending on thepaper material. These volumes would not be an excessive amount of fluidto pass through an integrated device for the purpose of removing FTA®chemistry from the FTA® papers. The following validate that the washedFTA® papers are able to support amplification.

Example 5 Isothermal Amplification of Target Nucleic Acids after Washingwith an Aqueous Buffer

Ten (10) ng of purified Mycobacterium tuberculosis (TB) genomic DNA wasapplied to a 1.2 mm FTA disc. Each disc was placed in separate 1.5 mltubes. Two hundred (200) μl of FTA Purification Reagent was added toeach tube, followed by incubation for 5 minutes at room temperature. Theadded FTA Purification Reagent was removed from the tube using a pipetteand discarded. The washing with FTA purification reagent was repeatedtwice, for a total of 3 washes with FTA Purification Reagent. Twohundred (200) μl of TE buffer with 0.01% Tween 20 (TET) was added to thetube containing washed genomic DNA on the FTA disc. The TET buffer inthe tube was incubated for 5 minutes at room temperature. The used TETbuffer from the tube was removed and discarded with a pipette. Thewashing of the genomic DNA with aqueous TET buffer was repeated once fora total of 2 washes with TET. The liquid was removed completely from thetube before transferring the FTA discs comprising the genomic DNA to thereaction tubes.

Isothermal Amplification by “Ping Pong”

The genomic DNA was adhered to the matrix, which was further amplifiedby “Ping Pong” amplification.

TB genomic DNA was used both in solution and on an FTA Classic card. TheFTA classic card was washed as described above. Both of the FTA classiccard containing genomic DNA after wash (washed) and before wash(unwashed) were subjected with amplification reaction mixture to amplifythe genomic DNA. 10× denaturation buffer (100 mM HEPES, pH 8, 1 mM EDTA,0.1% (v/v) Tween 20, 10 mg/ml BSA) was used before amplification. 10×Ping Pong Reaction Buffer (150 mM HEPES, pH 8, 30 mM MgCl₂, 1 mM MnSO4,0.1% Tween 20, 2.5 mM dATP, 2.5 mM dCTP, 2.5 mM dGTP, 5 mM dTTP, 50 mM(NH4)₂SO₄, 10 mM TCEP) was used for Ping Pong amplification reaction. TB15 oligo mixture was used for the amplification reaction; the sequencesof the primers are listed below. Twenty (20) pmoles of each primer wasused for each of the reactions.

TABLE 2 Sequence list of the primers used ID Sequence Name SEQ IDCAT GAA GTG CTG GAA GGA T/ LRS1 NO: 1 ideoxyI/*C SEQ IDTCC TCT AAG GGC TCT CGT T/ LRS2 NO: 2 ideoxyI/*G SEQ IDAAA TTA TCG CGG CGA ACG G/ LRS3 NO: 3 ideoxyI/*C SEQ IDGGCAG ATT CCC GCC AGA/ TB NO: 4 ideoxyI/*C F60 SEQ IDAAA ACA GCC GCT AGT CCT A/ TB NO: 5 ideoxyI/*T F61 SEQ IDTC GCC CGC AAA GTT CCT C/ TB NO: 6 ideoxyI/*A F62 SEQ IDCC AAA CCG GGT CTC CTT C/ TB NO: 7 ideoxyI/*C F63 SEQ IDTAA GCT GCG CGA ACC ACT T/ TB NO: 8 ideoxyI/*A F64 SEQ IDCTG GGT TGA CAT CAC CCC/ TB NO: 9 ideoxyI/*C R66 SEQ IDAAG TCC TCG ATC GGA GAC A/ TB NO: 10 ideoxyI/*C R68 SEQ IDGAC AAC GAC ATC GAC CCG/ TB NO: 11 ideoxyI/*A R69 SEQ IDCGT CGA AAC GAG GGT CAG A/ TB NO: 12 ideoxyI/*A R70 SEQ IDCTC GTC GAC GGG TGC CTT/ TB NO: 13 ideoxyI/*A R71 SEQ IDGTA CGT CAT GTC CTT GTC TTT/ LRS NO: 14 ideoxyI/*C 4 SEQ IDTCA CCG GTG TTG TTG TTG AT/ TB NO: 15 ideoxyI/*A LRS6

The reactions were prepared as shown in Table 3. After mixing thereactants, the reaction mixtures were heated at 95° for 2 minutes fordenaturation, followed by incubated on ice. The aliquots of 14.84 μlwere used further for each amplification reaction.

TABLE 3 The reaction protocol for denaturation: (volumes are in μl) RxnID Reactant A B G H 10x Denaturation Buffer 6 4 2 2 TB gDNA, 50 ng/μl(Soln) − 0.4 − − Washed 1.2 mm Punch − − + − Unwashed 1.2 mm Punch − −− + TB 15 Oligo Mix 6 4 2 2 Ethylene Glycol 12 8 4 4 99.5% Formamide0.75 0.5 0.25 0.25 Water 19.77 12.78 6.59 6.59 Total Volume 44.52 29.6814.84 14.84

To the above aliquots, 1/10^(th) volume of 10× reaction buffer, 81.6units Bst DNA polymerase large fragment, two microgram E coli SSB, 0.5micrograms E coli EndoV Y80A mutant were added. After addition, thereaction mixtures were incubated at 45° C. for 60 minutes. Supernatantsof the reactions were loaded on a 15% Acrylamide TBE-Urea Gel andvisualized by SYBR gold staining.

The gel electrophoresis analysis is shown in FIG. 8A. Lanes 1, 2, 3, 4and 5 were molecular weight marker, no template control, amplificationreaction of genomic DNA in a solution, amplification reaction of genomicDNA on a substrate after extensive wash, and amplification reaction ofgenomic DNA on a substrate without washing, respectively. The reactionmixtures of A, B, G and H (from the above denaturation) were used foramplification reaction and loaded to the gel, as described in Table 4below. Table 4:

TABLE 4 Reactions loaded to gel Lanes Reaction mixture 1 10 Base Ladder2 Rxn A, No Enzyme, No Template 3 Rxn B + Enzyme + Solution Template 4Rxn G + Enzyme + Washed Punch 5 Rxn H + Enzyme + Unwashed Punch

The substrate was washed with an aqueous buffer, not with ethanol. Theresults (FIG. 8A) indicated that the genomic DNA which was loaded on thesample application zone was not washed out during five repeated washingsteps with an aqueous buffer (lane 4—washed punch) and was amplified bythe “Ping Pong” isothermal amplification reaction. The unexpectedfinding was that after washing with an aqueous buffer, the DNA was stillattached to the substrate and amplified as similar as it was amplifiedin a solution (lane 3). In addition, the amount of amplification productgenerated for washed DNA (lane 4) was similar to reactions performed onthe equivalent amount of DNA in solution (lane 3). Unlike target DNA,the amplification product was not immobilized on the substrate. Theamplification product was eluted out from the substrate and collected asa supernatant. The supernatant was loaded to the gel for furtheranalysis. The gel is marked as “expected product” in FIG. 8 A, as theproduct molecular weight was estimated from the known primer sequence.

Amplification of DNA on a Substrate Using UStar FASTeasy AmplificationKit

An amplification of DNA attached to substrate was also performed by“UStar FASTeasy” kit. The UStar FASTeasy TB Isothermal AmplificationDiagnostic Kit was purchased from UStar Biotechnologies (Hangzhou),Ltd., China. TB genomic DNA, both in solution and on an FTA Classiccard, washed and unwashed samples were used for amplification reaction.The reaction mixtures were prepared as described in Table 5 below:

TABLE 5 Reaction scheme for UStar Fasteasy amplification: ID Reactionmixture Lanes I +Enzyme, No Template 1 J +Enzyme, +Solution Template 2 K+Enzyme, +Washed Punch (with Template) 3 L +Enzyme, +Unwashed Punch(with Template) 4

The reactions were set up as indicated in Table 6 below. Theresuspension buffer was added to the dried pellets of the amplificationreaction mixture and incubated at room temperature for 2-3 minutes tocompletely dissolve the reagents. The genomic DNA or substrate punchcontaining genomic DNA was added to the reaction mixture and incubatedthe reactions at 63° C. for one hour. Reactions were stopped by theaddition of 2 μl of 210 mM EDTA.

TABLE 6 Reaction protocol for UStar Fasteasy amplification (volumes arein μl) ID Reactants I J K L Resuspension Buffer 15 15 15 15 Water 4 3 44 TB gDNA (10 ng/μl) − 1 − − Total Volume 19 19 19 19

The reactions (Table 5) were analyzed by gel electrophoresis on 15%Acrylamide TBE-Urea Gel and visualized by SYBR gold staining From theabove reaction mixtures, 3 μl of the supernatant from the reactionmixtures were loaded to the gel for analysis, wherein 10 ng of DNA waspresent in the sample (load). The reactions were loaded to 15%Acrylamide gel in lanes 1, 2, 4 and 5 as shown in Table 5 and FIG. 8B.The result as shown in FIG. 8 B, is indicated that the genomic DNA whichwas loaded on the sample application zone of the substrate punch was notwashed out during five repeated washing steps with an aqueous buffer andwas amplified (lane 4—washed punch) by the “UStart FASTeasy” isothermalamplification reaction. In addition, the amount of amplification productgenerated is similar to reactions performed on the equivalent amount ofDNA in solution (lane 2), whereas no template control was loaded to lane1, molecular weight marker was loaded to lane 3 and unwashed punch wasloaded to lane 5. Unlike target DNA, the amplification product was notimmobilized on the substrate and was eluted out in the supernatant. Thegel is marked as “expected products” in FIG. 8 B, as the productmolecular weight was estimated from the known primer supplied with thekit.

Amplification of DNA on a Substrate Using “Helicase DependentAmplification” or “HDA”

The DNA attached to the substrate was further amplified using “HDA”amplification kit, IsoAmpIII Universal tHDA Kit (catalog # H0120S) waspurchased from Biohelix Corp. TB genomic DNA, both in solution and on anFTA Classic card, washed and unwashed samples were used foramplification reaction. The reaction mixtures were prepared from bulkmix, as described in Table 7 below:

TABLE 7 Reaction scheme for HDA amplification: Bulk mix (volumes are inμl) ID Reactants 1x 16x 10X Annealing Buffer II (came w/kit) 5 80 100 mMMgSO4 2 32 500 mM NaCl 4 64 IsoAmp dNTP Soln 3.5 56 Forward Primer(supplied in kit) 0.75 12 Reverse Primer (supplied in kit) 0.75 12 TotalVolume 16 256

The amplification reaction mixture was prepared from the above bulkmixture (Bulk mix) as described in Table 8, below:

TABLE 8 Reaction mixture for HDA amplification: (volumes are in μl)lanes Reactants 1 2 4 5 Water 32 31 32 32 Bulk Mix 16 16 16 16 Template(liquid, 10 ng/μl) −  1 − − Template (1.2 mm punch, − − + − washed)Template (1.2 mm punch, − − − + NOT washed) IsoAmp III Enzyme Mix  2  2 2  2 Total Volume 50 50 50 50

The reaction mixtures were mixed thoroughly and incubated for 90 minutesat 65° C. and then stopped each reaction by adding 2 μl of 0.5 M EDTA.The supernatants from the above reactions were analyzed by gelelectrophoresis. The supernatant of 3 μl was loaded on 15% AcrylamideTBE-Urea Gel and visualized by SYBR gold staining, wherein lane 1 is notemplate control, lane 2 is amplification in solution, lane 3 ismolecular weight market, lane 4 is template amplified on washed punch,and lane 5 is template amplified on unwashed punch. Results are shown inFIG. 8C, wherein the data indicated that the genomic DNA that was loadedon the sample application zone was not washed out during 5 repeatedwashing steps with an aqueous buffer. Further, the attached target DNAof the substrate was capable of being amplified by the Helicasedependent amplification reaction. In addition, the amount ofamplification product generated in washed punch (lane 4) is similar toreactions performed on the equivalent amount of DNA in solution (lane2). Unlike genomic DNA, the amplification product was not immobilized onthe substrate. The amplification product was eluted from the substratewas in supernatant, wherein the genomic DNA was still attached to thesubstrate. The amplification product was loaded to the gel as asupernatant. The gel is marked as “expected product” in FIG. 8 C, as theproduct molecular weight was estimated from the known primer suppliedwith the kit.

Example 6 Lateral Flow Migration of High Molecular Weight DNA (≧50 kb)and Effect of Washing

To examine the migration of the template DNA during the washing andamplification processes, approximately one million cells in a 10 μlvolume (10⁶ CFU) from an overnight culture of methicillin-sensitiveStaphylococcus aureus (S. aureus; ATCC) or MSSA were applied to thetreated GF/F square (step 1, 52 of FIG. 9 A) and were then heated at 49°C. for 10 minutes to lyse the cells. An amplification reaction buffercomprising various reactants was used for washing solution, wherein theamplification buffer is devoid of enzyme, such as polymerase.

Lateral flow was initiated 54 by placing amplification reaction mixtureon the pointed tip of the strip (substrate) 12, as shown in step 2 (54)of FIG. 9 A. Subsequently amplification buffer (without enzymes) wasallowed to flow through the strip. 25 μl of amplification reactionmixture were added to the strip 12 without a wicking pad, while 100 μlof amplification reaction mixture were added to the strip 12 with awicking pad, wherein the amplification mixture did not include enzymes.Capillary flow was allowed to continue for 30 minutes at 49° C. Oncecapillary flow was terminated, each strip was sectioned into five equalportions as indicated in step 3 (56) of FIG. 9 A.

Similar sections from two strips were treated as follows: 1) eachsection was transferred to a separate microcentrifuge tube, 2) 200 μl ofTE Buffer, pH 8 (Life Technologies) was added to each tube and thenvortexed, 3) the TE Buffer was recovered and further added to separatemicrocentrifuge tubes and centrifuged at 16,000×g for 10 minutes topellet intact cells and debris, 4) each pre-cleared portion of TE Bufferwas added back to the appropriate washed porous matrix and heated to 95°C. for 10 minutes, 5) the TE Buffer was recovered into separatemicrocentrifuge tubes and the nucleic acid in each tube precipitatedusing the DNA Extractor® Kit (Wako Chemicals, USA) according to themanufacturer's instructions, wherein as an exception, Proteinase K wasomitted and 6) each pellet was resuspended and analyzed by gelelectrophoresis through a 1% agarose gel in TBE Buffer (Affymetrix-USB).Following electrophoresis, the gel was stained with SYBR Gold (LifeTechnologies) and imaged using a Typhoon™ Variable Mode Imager (GeneralElectric Company), which is shown in FIG. 9 B. As illustrated in FIG. 9B, the substantial amount of genomic DNA stays in the glass fiber pad(lane 2) even after significant washing (100 μl). So, washing of targetnucleic acids with an amplification buffer (without enzyme) does notresult migration of the target nucleic acids on the substrate (lanes 3,4, and 5) for both cases, the substrate with wick and without wick. Theimage shows that after diffusion of the amplification buffer and the TEbuffer, most of the genomic DNA (molecular weight>50 kb) were positionedaround the first end 11 (segment 2, FIG. 9 B) of the substrate (FIG. 6).

For gel electrophoresis, each small piece was placed into a separatedsample well of 0.8% agarose gel. Electrophoresis was carried out with avoltage of 120 V for 45 minutes. After electrophoresis, the agarose gelwas stained by 1xSYBR Green I solution for 1.5 hours and then visualizedusing a Typhoon™ Trio™ variable mode imager, GE Healthcare, New Jersey,USA. The Quick-Load(i) 1 kb DNA Ladder (New England BioLabs) was used asa DNA marker. Quick-Load(i) 1 kb DNA Ladder was a premixed,ready-to-load molecular weight marker containing bromophenol blue as atracking dye. The DNA ladder consists of 10 bands: 10 kb, 8 kb, 6 kb, 5kb, 4 kb, 3 kb, 2 kb, 1.5 kb, 1 kb and 500 bp.

Example 7 Lateral Flow Migration of Low Molecular Weight DNA Amplicons(≦50 kb)

An isothermal amplification kit was obtained from Epoch Biosciences,Inc. (Bothell, Wash.) containing primers specific for the LDH1 gene ofmethicillin resistant S. aureus (MRSA). Genomic DNA for MRSA wasobtained through NIH Biodefense and Emerging Infections ResearchResources Repository, NIAID, NIH. Genomic DNA from Staphylococcusaureus, Strain HFH-29568, NR-10314 was also used. A 5 mm diameter punchwas obtained from an unmodified GF/F porous matrix (Whatman® GEHealthcare).

Twenty-three (23) nanograms of MRSA genomic DNA were added to the 5 mmpunch and the punch was immediately assembled on a lateral flow strip 12containing a wicking pad as found in Example 1 (as shown in FIG. 8) andalso showed in FIG. 10 A. Lateral flow was initiated by applying fifty(50) microliters of amplification reaction mix containing enzymes on thepointed tip and the flow continued for 30 minutes at 49° C. At the endof the incubation period, the strip 12 was sectioned into seven (7)equal portions of 5 mm each as indicated in the work flow of FIG. 10 A.

The liquid remaining in each 5 mm section of the strip was isolated bycentrifugation and a portion analyzed by electrophoresis through a 15%acrylamide TBE urea gel (Life technologies). Following electrophoresis,the gel was stained with SYBR Gold (Life Technologies) and imaged usinga Typhoon™ Variable Mode Imager (General Electric Company). FIG. 10 Bdemonstrates (in a dotted lined box) that the expected amplificationproducts are found in lanes 0, 1, 2, 3, 4, 5 and 6, with some appearingin the wick (lane W) compared to the gel loaded with no template control(FIG. 10 C), wherein no amplification product was determined Example 5,FIG. 9 B demonstrated that the genomic DNA did not migrate duringlateral flow, and this Example 7 showed increasing amounts ofamplification products in each of the sections of 0 to 6 of FIG. 10 B,which established that one or more isothermal amplification reactions ofMRSA DNA occurred during lateral flow, while the amplicons with lowermolecular weights are determined by DNA gel electrophoresis (FIG. 10 B).The reactions were also executed in a tube with template and in anothertube without template under the same conditions, as shown tube (+) andtube (−) respectively in both of the FIGS. 10 B and 10 C. A light bandof genomic DNA was observed in lane 0 (above the lane numbers) and inlane for tube +. The absence of this band of genomic DNA in lanes 1 to 6further established that the genomic DNA does not move with theamplicons during lateral flow.

Example 8 Amplification in-Flow Topology: Lateral Flow Separation ofHigh Molecular Weight DNA and Low Molecular Weight DNA

Efforts were made to evaluate amplification-in-flow topologies. Aschematic representation of the amplification-in-flow is shown in FIG.10 A.

Nitrocellulose grafted with poly(ethylene glycol) monomethyl ethermethacylate (PEGMA) or NC-PEG (Pegylated Nitrocellulose) was used forthis example). Polyethylene glycol methyl ether methacrylate (PEGMA) 300grafted nitrocellulose (NC-PEG) and PEGMA 300 grafted 903 cellulosepaper were fabricated by soaking the appropriate base substrate (FF60nitrocellulose or 903 cellulose) in an aqueous solution containing 10%(w/v) PEGMA 300 (Sigma-Aldrich) and 30% (v/v) Tween 20 (Sigma-Aldrich).Excess solution was removed and the treated matrices were subjected toE-beam (AEB, Advanced Electron Beam, e-Beam unit, EBLAB-150), withoperation voltage of 125 kV, and electron dosage delivery of 10 kGy.Following irradiation, the modified matrices were washed in distilledwater by orbital rotating for 30 minutes. The washing steps wererepeated three times. The membranes were then allowed to air dry at roomtemperature overnight.

An isothermal amplification kit was obtained from Epoch Biosciences,Inc. (Bothell, Wash.) containing primers specific for the LDH1 gene ofmethicillin resistant S. aureus (MRSA). Genomic DNA for MRSA wasobtained through NIH Biodefense and Emerging Infections ResearchResources Repository, NIAID, NIH. Genomic DNA from Staphylococcusaureus, Strain HFH-29568, NR-10314 was also used. Purified MRSA DNA (10⁶copies) was spotted onto a glass fiber pad (GF/F) bridging a gap in anNC-PEG strip. The strip was then placed into a conical tube with 50 μlof isothermal DNA amplification reaction buffer and sealed. The tube wasincubated at 49° C. for 30 minutes while the isothermal DNAamplification reaction buffer flowed through the substrate (strip ortest strip) and target DNA started amplified. Subsequently, the teststrip was removed and cut into 5 mm segments that were then centrifugedto recover the products in each segment and analyzed via gelelectrophoresis. FIG. 10 B illustrated that, below the sample pad(position −1), there is no signal in the gel, whereas on and beyond thesample pad for positions 0 through 6, the amount of amplicons increasesas the distance from the sample pad and subsequently amplification timeincreases (the amplification products are indicated in the dotted linedbox, FIG. 10 B). The band from the sample pad (position 0) appears moreintense due to more product volume was recovered from the glass fiberthan the NC-PEG (approximately 40% fold higher). This resultdemonstrates amplification-in-flow and highlights the utility of glassfiber materials when placed upstream of the flow path. When using S.chromogenes, it was observed that the template nucleic acids eluted fromGF-F Elute with slower apparent kinetics when compared withcellulose-based FTA Elute. The delay may contribute toamplification-in-flow efficiency.

Example 9 Detection of Amplicons on the Substrate

An isothermal amplification kit was obtained from Epoch Biosciences,Inc. (Bothell, Wash.) containing primers specific for the LDH1 gene ofmethicillin sensitive S. aureus (MSSA). Approximately one million cellsin a 10 μl volume (10⁶ CFU) from an overnight culture ofmethicillin-sensitive Staphylococcus aureus (S. aureus; ATCC) or MSSAwere applied by pipet (or a swab dosed with MSSA) onto a glass fibersample pad 62 (GF/F, containing the FTA® Elute chemistry) bridging a gapin an NC-PEG strip 12 with a test line 20 and a wick 28 (FIG. 11 A). Insome examples, the test strip had an additional glass fiber pad (GF/F)21 placed on top of the strip before the test line 20 that could beremoved after amplification for analysis. The strip 64 was then heatedat 49° C. for 15 minutes to facilitate faster lysis of the MSSA cells.After lysis, the strip was placed into a conical tube with 100 μl ofisothermal DNA amplification (iSDA) reaction buffer and sealed 66. Thestrip was then incubated at 49° C. for 30 minutes as the isothermal DNAamplification reaction buffer flowed through the strip, simultaneouslypurifying the DNA, rinsing residual FTA® Elute chemistry from the glassfiber application site, and amplifying the ldh1 locus parallel. Theamplification in-flow was estimated which started after 25-50 μl ofisothermal DNA amplification reaction buffer entered the strip 12.

Following amplification-in-flow, the strip was transferred to a secondconical tube to deliver 100 μl of a “chase” buffer solution comprised ofcontaining streptavidin coated blue polystyrene beads to that will bindto the biotin labeled probes and enable colorimetric detection of ldh1upon capture at the test line. As shown in FIG. 11 B, MSSA-dosed strips(including designs both with and without the optional glass fiber pad)showed positive results with an arrow corresponding to the bands, bothin 72 and 74, while the no-template control sample without MSSA cellsshowed negative result 70. Similar MSSA examples repeated withcellulose-based FTA Elute rather than GF-F Elute, showed less robustperformance (little to no test line development for ldh1), whichdemonstrates that amplification in place is slower and less efficientwhen using upstream cellulose materials.

The above examples demonstrated the ability of the substrate tointroduce a sample directly to the substrate, lyse the cells, purify theDNA, amplify in-flow, and detect the target in a one-dimensional lateralflow device.

While the disclosure has been illustrated and described in typicalembodiments, it is not intended to be limited to the details shown,since various modifications and substitutions may be made withoutdeparting in any way from the spirit of the present disclosure. As such,further modifications and equivalents of the disclosure herein disclosedmay occur to persons skilled in the art using no more than routineexperimentation, and all such modifications and equivalents are believedto be within the spirit and scope of the disclosure as defined by thefollowing claims.

What is claimed is:
 1. A method comprising: applying a sample comprisingtarget nucleic acids to a sample application zone of a substrate;applying an aqueous buffer to the sample application zone of thesubstrate for washing away or diluting one or more inhibitors present onthe sample application zone; and applying an isothermal nucleic acidamplification reaction mixture to the sample application zone to amplifythe target nucleic acid to form a nucleic acid amplification product;wherein the target nucleic acid having a first molecular weight issubstantially immobilized at the sample application zone and theamplification product has a second molecular weight.
 2. The method ofclaim 1, further comprising contacting the sample with a lysis reagent.3. The method of claim 2, wherein the sample comprises the lysisreagent.
 4. The method of claim 2, wherein the lysis reagent isimpregnated in the sample application zone.
 5. The method of claim 2,wherein applying the aqueous buffer prior to application of theisothermal nucleic acid amplification reaction mixture washes or dilutesthe lysis reagent from the substrate.
 6. The method of claim 1, whereinthe aqueous buffer is a solvent of the nucleic acids.
 7. The method ofclaim 1, wherein the aqueous buffer is at least one oftris(hydroxymethyl)aminomethane and ethylenediaminetetraacetic acid (TE)buffer, and PBS or TE in which the tris buffer is substituted withHEPES.
 8. The method of claim 1, wherein applying the isothermal nucleicacid amplification reaction mixture washes the lysis reagent from thesubstrate.
 9. The method of claim 1, wherein applying the isothermalnucleic acid amplification reaction mixture washes away one or moreinhibitors present on the sample application zone.
 10. The method ofclaim 1, wherein the first molecular weight is in a range of at leastabout 50 kb.
 11. The method of claim 1, wherein the first molecularweight is in a range from about 50 kb to about 150 kb.
 12. The method ofclaim 1, wherein the nucleic acids are DNA, RNA or a combinationthereof.
 13. The method of claim 1, wherein the substrate is made ofcellulose membrane, a nitrocellulose membrane, modified porousnitrocellulose or cellulose based substrates,polyethyleneglycol-modified nitrocellulose, a cellulose acetatemembrane, a nitrocellulose mixed ester membrane, a glass fiber, apolyethersulfone membrane, a nylon membrane, a polyolefin membrane, apolyester membrane, a polycarbonate membrane, a polypropylene membrane,a polyvinylidene difluoride membrane, a polyethylene membrane, apolystyrene membrane, a polyurethane membrane, a polyphenylene oxidemembrane, a poly(tetrafluoroethylene-co-hexafluoropropylene) membrane,or a combination thereof.
 14. The method of claim 1, wherein thesubstrate is a quartz matrix.
 15. The method of claim 1, wherein thetarget nucleic acid is not heat denatured or chemically denatured priorto or during amplifying the target nucleic acid to form a nucleic acidamplification product.
 16. The method of claim 1, wherein the secondmolecular weight of the amplification product is lower than about 50 kb.17. The method of claim 1, wherein the amplification product is notsubstantially immobilized at the sample application zone.